What are the differences between these microbial ecosystems? WWwwe now know that, like other org gm,anisms, bacteria exhibit social behaviors. Bacterial cell escaping for a rare moment of peace and quiet contemplation Sociomicrobiology I. Cell signaling A. Definition/description B. Intraspecific (within spp.): Myxococcus C. Interspecific (between spp.): Pseudomonas aureofaciens II. Biofilms A. Biofilm formation B. Planktonic cells vs. biofilm cells C. General characteristics, structures D. Biofilms as social entities Small diffusible molecules mediate bacterial communication O O N H AHL O O O N H O http://www.ted.com/index.php/talks/bonnie_bassler_on_how_bacteria_communicate.html Why cell-cell signaling in bacteria? Often, single cells in a population might benefit from knowing how many cells are present… “A multitude of bacteria are stronger than a few, thus by union are able overcome obstacles too great for few.” -- Dr. Erwin F. Smith, 1905 (Father of Plant Bacteriology) Pseudomonas aeruginosa in lungs Xylella fastidiosa in xylem Why cell-cell signaling in bacteria? Cell-cell signaling enables bacteria to coordinate behavior to respond quickly to environmental stimuli… such as: -presence of suitable host -change in nutrient availability -defense/competition against other microorganisms -many others! CmmiCommunica tion among btbacteri i:a: The example of Myxococcus xanthus, the Wolf Pack feeder of bacteria http://cmgm.stanford.edu/devbio/kaiserlab/about_myxo/about_myxococcus.html Cooperation among cells in a population: myxobacteria. The myxobacteria are Gram-negative, ubiquitous, soil-dwelling bacteria that are capable of multicellular, social behaviour. In the presence of nutrients, “swarms” of myxobacteria feed cooperatively by sharing extracellular digestive enzymes, and can prey on other bacteria. When the food supply runs low, they initiate a complex developmental program that culminates in the production of a fruiting body composed of hundreds of thousands of cells. The myxobacteria communicate with each other, and coordinate their movements through a cell- contact-dependent signal. --Adapted from Dale Kaiser, 2003. Nat. Rev. Microbiol. 1:45 Stigmatella aurantiaca Myxococcus stipitatus Stigmatella aurantiaca PHOTOS COURTESY OF HANS REICHENBACH Myxococcus social behavior Coordinated movement, unlike random walk of chemotaxis -slime layer contains fibrils -slime and fibrils are “wrapped” around all cells -pilus of one cell anchors on fibril of another -retraction of pilus = “pulling” -called “S motility”, named for slime trails Social Motility -Also indeppyendent, or “A motility”, for Adventurous Motility Five signals necessary for fruiting body formation -A signal: mix of 6 amino acids at low concentration -C factor: for “contact” signal -membrane-bdbound proteins at cell poles -B, D, and E: remain unknown, but mutants can be restored to normal fruiting body formation by extracellular complementation -guide group from one developmental stage to next -A starts signaling cascade, then C produced. C autoinduces till enough cells are swarming to form fruiting bodies. Swarming: M. xanthus coordinates cell movement with C-factor Gliding mo tility in M. xa nthusinvo lve s two differe nt “gliding ma chine s”, one at each cell pole: 1. the S-machine, which depends on type IV pili 2. the A-machine, which seems to involve a slime extrusion mechanism. C-factor is a small (20 kD), membrane-bound protein NOT diffusible requires cell-cell contact autoinduces, there are thresholds of C-factor for each subsequent developmental stage C-factor organizes the movement of cells: rippling aggregation end-to-end packing in “rafts” inside spores C-factor increases rate of cell travel by: increased gliding speeds JM Kunder and D. Kaiser, 1982 longer gliding intervals decreased stop and reversal frequencies Spore formation: M. xanthus signals starvation with A-factor Cells sense that nutrient density is getting low, and release A-factor A-factor JM Kunder and D. Kaiser, 1982 -mix of 6 amino acids (result of proteolysis): trp, pro, phe, tyr,leu, ile -amino acids are 10-fold less than concentration necessary to support growth -only released by starving cells -each cell only releases a fixed amount of A-factor -diffusible If enough cells release A-factor, the population “agrees” to commit to fruiting body development, and begins the first stages of aggregation. There’s a time limit for fruiting body formation. During starvation, cells “sense impending doom” and seem to act proactively to fruit and disperse. (Why?). Cells sense actual starvation as the lack of one or more amino acylated tRNAs, which triggers the “stringent response” during which protein synthesis is temporarily shut down, so no fruiting body could be formed. Microbes are useful for studying ecological interactions and evolution… Fruiting bodies are not selected for in abundant nutrients: Myxobacteria share characteristics of uni- and multi-cellular organisms. They grow and divide as do all Gram-negative bacteria. In abundant nutrients, they don’t form fruiting bodies. In vitro natural selection: grew 12 “lines” of Myxococcus in test tube without starving, for 1000 generations. More than half of these developed mutants that lost fruiting body development and sporulation efficiency. (Velicer, Kroos, LkiLenski, 1998. PNAS 95: 12376) Why is social behavior selected for, in the wild (in the soil)? 1. Spore is bigger than a cell: 0.2 mm in diameter. Can adhere to passing animal (e.g. worm, mite) and be carried to new food source. 2. Wolf pack fee ding : once spores germinat e in new spo t, the cells secre te extracellular enzymes en masse, thus degrading food much more efficiently Like M. xanthus, which uses A-factor to coordinate sporulation, many species coordinate cell-cell social behaviors through quorum sensing. O O N H O LuxR LuxR AHL AHL (lux box:) ACCTGTAGGATNGTACAGGT But, can different species of bacteria communicate with one another? Cross-communication among rhizosphere bacteria: The example of Pseudomonas aureofaciens and its neighbors Lawn of P. aureofaciens 30-84I (PhzI-) ‘Good Neighbors’ Positive communication ‘Bad Neighbors’ Negative Communication Lawn of P. aureofaciens 30-84 (WT) Sociomicrobiology I. Cell signaling A. Definition/description B. Intraspecific (within spp.): Myxococcus C. Interspecific (between spp.): Pseudomonas aureofaciens II. Biofilms A. Biofilm formation B. Planktonic cells vs. biofilm cells C. General characteristics, structures D. Biofilms as social entities Bacteria in liquid culture = “planktonic” -used to study most microbial phenomena prior to 1990’s -used to descr ibe quorum sensing (pro ba bly inaccura te ) Bacterial climax communities are “biofilms” -communities o f microbes associated with a s ur face , t ypically encased in extracellular matrix -liquid/solid interface -air/water interface -sometimes no obvious interface (suspended aggregates) Biofilms are the “norm” and planktonic cells the exception in nature. Biofilm gene expression differs up to 70% from planktonic cells. Whoops, we’ve been studying the wrong thing all these years! Sauer et al., 2007. Microbe 2(7): 347 You’ve never seen a biofilm before? Yes you have… the plaque on your teeth, the sludge in your bathroom sink drain, the slime on your shower curtain… Epifluorescence microscopy of biofilm samples taken from shower curtains. These communities can contain diverse populations of microbes. (Micrograph from Appl. Environ. Microbiol. 70:4187-4192.) Fluorescently tagged bacteria in a biofilm. Notice water channels and extracellular matrix (e.g. polysaccharide). Stages in Biofilm Development 1. Attachment Sauer et al., 2007. Microbe 2(7): 347 2. Aggregation and growth into microcolonies (mediated by HSLs) 3. Maturation of biofilm -biomass and thickness governed by HSLs -rhamnolipid surfactants maintain water channels 4. Maintenance of biofilm -biofilms may “pump” water by changing ionic strength of milieu -dispersal -programmed cell death, releases nutrients and DNA for survivors (nutrition, transformation/HGT) Regulation of normal biofilm formation from planktonic cells Various of these genes (below) required, depending on species… no “core regulator” common to all species for biofilm formation has been identified. Chemotaxis genes Flage llar genes Alginate genes Sigma factors (RpoN, RpoS) Membrane transport proteins Membrane sensor proteins (GacA/S) Quorum sensing genes (LasR, RhlR) Signal genes (cyclic di-GMP) Biofilm formation is not a “stress response”. For example, the genes for biofilm formation are not the same as those that stimulate fruiting body/spore formation (induced by stationary phase sigma- factor). Bacteria in biofilms exhibit different physiology than planktonic cells. Environmental context - the following processes occur at different rates in the presence of plan kton ic vs. bifilbiofilm cells: C cycling and nutrient cycling Chemical reactions in bioreactors TiToxic chilhemical degra dtidation Corrosion of metal surfaces Medical context – biofilms are seen in 65 to 80% of all infections treated in the developed world. Biofil ms are to leran t to 1000X high er leve ls of antibio tics, phage, antibo dies, and antimicrobial peptides than those required to decimate populations of planktonic cells. WHY? A. EPS limit s diffus ion or che la tes certa in compounds B. Different physiological states = differential resistance (exponential, stationary, dormant) -Adaptive stress responses make cells more resistant -Persister cells (dormant = target bound by antibiotic